Figure 4

Using biotin-labeled K19 aptamer to enrich its target protein. (a). Effect of trypsin pre-treatment on binding of aptamers to NB4 cells. NB4 cells were treated with trypsin at 37°C for 10 min. The binding of selected aptamers with trypsin-treated NB4 cells is illustrated: JH6 (a1); JH19 (a2); and K19 (a3). The final concentration of these aptamers in binding buffer was 300 nM. The background binding was measured by using biotinylated single-stranded negative control DNA. (b). Binding competition of biotinylated-K19 to NB4 cells by the unlabeled K19 aptamers. NB4 cells were pre-incubated with the unlabeled K19 aptamers (300 nM) for 1 hr prior to binding of the biotin-labeled aptamers. The fluorescence intensity of bound aptamers is shown by the histograms (blue, binding of biotinylated K19; red, binding of biotinylated K19 after blocking by non-labeled K19). The background binding was measured by using biotinylated single-stranded negative control DNA. (c). Silver-stained polyacrylamide gel electrophoresis (SDS-PAGE) separating the proteins captured by aptamer K19. NB4 cells were pre-incubated with or without the unlabeled aptamer K19 for 1 hr prior to binding of the biotin-labeled aptamers. The NB4 cells were then lysed. The protein-aptamer DNA complex(s) were captured by the magnetic streptavidin beads, and were then separated by SDS-PAGE followed by silver staining for detection of characteristic protein bands. Lane M, molecular markers; Lane 1, proteins captured with streptavidin beads (no aptamer); Lane 2, proteins captured by biotin-aptamer K19 after blocking by unlabeled aptamer K19; Lanes 3 and 4, protein captured with biotinylated aptamer K19.